The long-term objective of this proposal is to elucidate the role of the glycerophosphocholine (GPC)-derived mediator Platelet-activating factor (PAF) in keratinocyte stress responses and cytotoxicity. Recent studies have demonstrated that many clinically relevant cytotoxic agents including ultraviolet B radiation (UVB) damage human keratinocytes in part by inducing cytokine production and oxidative stress. Severe UVB damage to human keratinocytes can also result in programmed cell death (apoptosis). Because PAF is produced by keratinocytes in response to oxidative stress and many pro-inflammatory cytokines, and keratinocytes express functional PAF receptors (PAF-R) linked to the production of many of the same cytokines associated with epidermal damage, the role of the PAF system in epidermal cytotoxicity has been a focus of our group. The original funded studies have created several novel model systems to study the epidermal PAF-R and have structurally characterized novel PAF-R agonists produced by epidermal cells in response to UVB. First, retroviral vector strategies were used to express a functional PAF-R in the PAF-R-negative epithelial cell line KB, and to ablate expression of the native PAF-R by an inducible antisense-PAF-R vector. In addition, functional xenografts of human keratinocytes overexpressing the PAF-R were made in SCID mice, and mice lacking a functional PAF-R were obtained. UVB irradiation of epidermal cells resulted in the production of both PAF and non-enzymatically produced GPC with sn-2 short-chained fatty acids (PAF-like lipids [PAF-LL]) that are potent PAF-R agonists. UVB-induced PAF-R agonistic activity is relevant since expression of the epidermal PAF-R in KB cells results in an augmentation of UVB-induced cytokine production and cytotoxicity in vitro. UVB irradiation of xenografts of keratinocytes overexpressing the PAF-R resulted in increased amounts of apoptosis over vector control xenografts. We hypothesize that oxidative damage to the keratinocyte induces PAF-R agonists that can augment cytokine production and cytotoxicity via activation of the epidermal PAF-R. The following three specific aims will test this by assessing the ability of the epidermal PAF-R to modulate UVB-induced cytokine production 1) in vitro; and 2) in vivo; and to define mechanistically the signal transduction pathways important in the PAF-R augmentation of UVB-induced cytokine production and epidermal cytotoxicity. These findings will aid in the understanding of the mechanism(s) by which oxidative stress can be involved in cutaneous pathophysiology.